Journal: bioRxiv

Article Title: Differential requirements for mitochondrial electron transport chain components in the adult murine liver

doi: 10.1101/2021.07.15.452466

Figure Lengend Snippet: (A) Overview of the pyrimidine synthesis pathway and its interaction with the mitochondrial electron transport chain. DHODH resides in the inner mitochondrial membrane and transfers electrons from dihydroorotate directly to coenzyme Q (CoQ), bypassing complex I. (B) Levels of DHODH in livers of the indicated genotype, assessed by Western blot. β2-microglobulin levels are shown as a loading control. (C) Relative abundance of pyrimidine synthesis pathway metabolites in livers of the indicated genotype. n=6 mice per group. The same color scheme is used throughout this figure. (D) Relative abundance of dihydroorotate and the orotate / dihydroorotate ratio in livers of the indicated genotype, following treatment with vehicle or brequinar (BRQ). n=3-4 mice per group. (E) NAD + / NADH ratios in livers of the indicated genotype, following treatment with vehicle or brequinar (BRQ). n=3-4 mice per group. Statistical significance was assessed using two-way ANOVA (C,D,E) with adjustments for multiple comparisons. All data represent mean +/− standard deviation from biological replicates. Source data is available in Figure 5 – source data file 1. Uncropped western blots are available in Figure 5 – source data file 2.

Article Snippet: Brequinar sodium was purchased from Tocris (6196).

Techniques: Membrane, Western Blot, Control, Standard Deviation

Journal: bioRxiv

Article Title: Differential requirements for mitochondrial electron transport chain components in the adult murine liver

doi: 10.1101/2021.07.15.452466

Figure Lengend Snippet: (A) Liver weight (normalized to body weight) from mice of the indicated genotype and treatment condition. n=3-4 mice per group. BRQ, brequinar. The same color scheme is used throughout this figure. (B) Fasting plasma glucose levels in mice of the indicated genotype and treatment condition. n=3-4 animals per group. (C) Circulating plasma markers (total protein, albumin, and total bilirubin) of liver function in mice of the indicated genotype and treatment condition. n=3-4 mice per group. (D) Plasma markers (ALT and AST levels) of liver damage in mice of the indicated genotype and treatment condition. n=3-4 mice per group. (E) Relative abundance of pyrimidine synthesis pathway metabolites in livers of the indicated genotype and treatment condition. n=3-4 mice per group. Statistical significance was assessed using two-way ANOVA (A,B,C,D,E) with adjustments for multiple comparisons. All data represent mean +/− standard deviation from biological replicates. Source data is available in - source data file 1.

Article Snippet: Brequinar sodium was purchased from Tocris (6196).

Techniques: Clinical Proteomics, Standard Deviation

Journal: International Journal of Biological Sciences

Article Title: Glycine Decarboxylase Regulates Renal Carcinoma Progression via Interferon Stimulated Gene Factor 3-Mediated Pathway

doi: 10.7150/ijbs.104458

Figure Lengend Snippet: Suppression of GLDC induces dNTPs depletion, resulting in ROS accumulation and leading to mitochondrial stress in RCC cells. (A) Quantification of dTTP, dATP, dCTP, and dGTP levels in ACHN and Caki-2 cells. CTL, control; shGLDC, knockdown of GLDC. Data are shown as the means ± SD ( n =3). (B) Representative micrographs of immunofluorescence staining for mitoSOX with 4',6-diamidino-2-phenylindole (DAPI) counterstaining. ACHN cells were treated with brequinar (Bre) 10 µM for 48 h. ImageJ is used for quantification of the fluorescence intensity. NT, non-treated cells. Data are shown as the means ± SD ( n =3). (C) Representative micrographs of immunofluorescence staining for mitoSOX with DAPI counterstaining in control (CTL) and GLDC knock-downed (shGLDC) cells. The quantifications of fluorescence intensity are on the right side. Data are shown as the means ± SD ( n =3). MitoSOX staining intensity in ACHN CTL and shGLDC cells was confirmed by FACS analysis. Data are shown as the means ± SD ( n =3). (D) Representative micrographs of immunofluorescence staining for mitochondria (anti-HSP60) and mitochondrial (mt) DNA nucleoids (anti-DNA). ImageJ was used to calculate the size of mtDNA nucleoids. Mito, mitochondria. p values were calculated using two-tailed unpaired Student t -test. * p < 0.05, ** p < 0.01, and *** p < 0.001.

Article Snippet: Brequinar (Bre) sodium was purchased from Tocris, Bristol, UK.

Techniques: Control, Knockdown, Immunofluorescence, Staining, Fluorescence, Two Tailed Test

Journal: International Journal of Biological Sciences

Article Title: Glycine Decarboxylase Regulates Renal Carcinoma Progression via Interferon Stimulated Gene Factor 3-Mediated Pathway

doi: 10.7150/ijbs.104458

Figure Lengend Snippet: GLDC regulates RCC cell proliferation by activating ISGF3 through the inhibition of dNTP synthesis. (A) Growth curve of ACHN cells treated with brequinar (Bre) 10 µM or without (NT) was assessed by CCK-8 assays. Data are shown as the means ± SD ( n =3). (B) Western blot of indicated proteins in ACHN cells treated with Bre 10 µM or 20 µM for 48 h. (C) qPCR analysis of ISGs in ACHN treated with Bre 10 µM compared to non-treated control cells (NT). Data are shown as the means ± SD ( n =3). (D) Representative micrographs of immunofluorescence staining for mitoSOX in indicated cells with or without the addition of each dNs 10 µM and EHNA 5 µM for 48 h. The fluorescence intensity was calculated by ImageJ ( n =3). (E) Cellular growth of ACHN control (CTL) and GLDC knock-downed cells (shGLDC) after the addition of each dNs 10 µM and EHNA 5 µM to inhibit of degradation of dATP in cell culture. Data are shown as the means ± SD ( n =3). (F) Western blot of indicated proteins in ACHN CTL and shGLDC cells 48 h after the addition of each dNs 10 µM and EHNA 5 µM. p values were calculated using two-tailed unpaired Student t -test. * p < 0.05, ** p < 0.01, and *** p < 0.001.

Article Snippet: Brequinar (Bre) sodium was purchased from Tocris, Bristol, UK.

Techniques: Inhibition, CCK-8 Assay, Western Blot, Control, Immunofluorescence, Staining, Fluorescence, Cell Culture, Two Tailed Test

Journal: bioRxiv

Article Title: Screening in serum-derived medium reveals differential response to compounds targeting metabolism

doi: 10.1101/2023.02.25.529972

Figure Lengend Snippet: (A) Schematic illustrating the synthesis of nicotinamide adenine dinucleotide (NAD+) through the de novo Preiss-Handler or salvage pathways. NAMPT: nicotinamide phosphoribosyl transferase. FK866 is an inhibitor of NAMPT. (B) Effect of the NAMPT inhibitor FK866 on the number of A549 cells cultured in RPMI or ftABS for 72 hr as determined by CellTiter-Glo. Data shown are normalized to the vehicle-treated control and are the mean ± SD from two technical replicates. Data are from the screen in . (C) Concentrations of nicotinamide, nicotinic acid (NA) and nicotinamide mononucleotide (NMN) present in RPMI or ftABS. ND: measured but not detected. (D) Relative viability of A549 cells treated with vehicle or FK866 in ftABS or in RPMI, with or without addition of 1 μM NA for 72 hr as determined by CellTiter-Glo. Data are normalized to the vehicle-treated control and are the mean ± SD from three technical replicates. Statistical test was performed using multiple unpaired t test (ns, not significant; ****p < 0.0001; *p < 0.05). (E) Schematic illustrating the de novo synthesis and salvage pathways to generate the pyrimidine nucleotide uridine monophosphate (UMP). DHODH: dihydroorotate dehydrogenase. Brequinar is an inhibitor of DHODH. (F) Effect of the DHODH inhibitor brequinar on the number of A549 cells cultured in RPMI or ftABS for 72 hr as determined by CellTiter-Glo. Data shown are normalized to the vehicle-treated control and are the mean ± SD from two technical replicates. Data are from the screen in . (G) Concentration of uridine present in RPMI or ftABS. (H) Relative viability of A549 cells treated with vehicle or brequinar in ftABS or in RPMI, with or without addition of 6 μM uridine for 72 hr as determined by CellTiter-Glo. Data are normalized to vehicle-treated control and are the mean ± SD from three technical replicates. Statistical test was performed using multiple unpaired t test (ns, not significant; ****p < 0.0001; **p < 0.01).

Article Snippet: Brequinar , Cayman Chemical Company , 24445.

Techniques: Cell Culture, Control, Concentration Assay

Journal: bioRxiv

Article Title: Screening in serum-derived medium reveals differential response to compounds targeting metabolism

doi: 10.1101/2023.02.25.529972

Figure Lengend Snippet:

Article Snippet: Brequinar , Cayman Chemical Company , 24445.

Techniques: Recombinant, Labeling, Clinical Proteomics, Generated, Cell Viability Assay, Software, Membrane

Journal:

Article Title: New Immunosuppressive Drugs: Mechanistic Insights and Potential Therapeutic Advances

doi:

Figure Lengend Snippet: Diversity and modes of action of “new” immunosuppressive drugs

Article Snippet: Brequinar sodium (quinoline carboxylic acid derivative) , Dupont-Merck Pharmaceutical Co., Wilmington, DE , No , Makowka & Cramer 1992 , 1993.

Techniques: DNA Synthesis, Activation Assay

Journal:

Article Title: New Immunosuppressive Drugs: Mechanistic Insights and Potential Therapeutic Advances

doi:

Figure Lengend Snippet: Structure of brequinar sodium (BQR) superimposed on a flow diagram to indicate the probable sites of action of the drug on pyrimidine biosynthesis. Sites of enzyme action are denoted by numbers in parentheses. In addition to inhibiting dihydroorotate dehydrogenase, our recent observations indicate that BQR also inhibits cytidine deaminase. The enzymes involved in the pathway are (1) dihydroorotate dehydrogenase; (2) orotate phosphoribosyltransferase, orotidine 5′– monophosphate decarboxylase, nucleoside monophosphate kinase and nucleoside disphosphate kinase (in sequence along the pathway); (3) nucleoside kinase; (4) cytidine deaminase and (5) CTP synthetase. CTP=cytidine triphosphate; UTP = uridine triphosphate.

Article Snippet: Brequinar sodium (quinoline carboxylic acid derivative) , Dupont-Merck Pharmaceutical Co., Wilmington, DE , No , Makowka & Cramer 1992 , 1993.

Techniques: Sequencing